Parkinson's disease, the second most common neurodegenerative disorder, is marked by progressive dysfunction and loss of nigral dopaminergic neurons. This cardinal feature is accompanied by the accumulation of protein inclusions in dopaminergic neurons and their processes (Lewy bodies and neurites), the major constituent of which is a-synuclein (aS). aS has 7 repeats resembling the lipid-binding a-helical domains of apolipoproteins, and its binding to phospholipid membranes markedly alters its secondary structure. We have recently: a) discovered homologies of aS with the fatty acid (FA) binding protein (FABP) family;b) found that pure aS binds free FAs reversibly;c) detected a pool of highly soluble, lipid-associated aS oligomers in dopaminergic cells, normal mouse and human brains and, at elevated levels, in PD and DLB brains;d) shown that exposure of living mesencephalic neurons to polyunsaturated FAs enhances ~ and to saturated FAs retards ~ the formation of soluble aS oligomers;and e) documented increased endogenous PUFA levels and membrane fluidity in aS-overexpressing neurons, and the opposite in aS knock-out mice. Based on these findings, we hypothesize that aS normally interacts with FAs in both the aqueous and membrane-phospholipid compartments of the cytoplasm and helps regulate aspects of lipid composition (particularly PUFA content) and thus membrane properties, and that aS-FA interactions help regulate the oligomerization of aS and can thus initiate aS assembly into first soluble and then insoluble oligomers. To pursue this molecular hypothesis about aS function and dysfunction, we now propose a series of interrelated goals. 1) To attempt to prove that altering endogenous PUFA levels (e.g., lowered in cells treated with a A6 desaturase inhibitor or elevated in mice modeling Zellweger's syndrome) induces corresponding decreases or increases in endogenous aS oligomers in brain cells. 2). To examine the effects of aS-FA interactions on the formation, ultrastructure and biophysical properties of membrane vesicles in living cells. 3) To ascertain whether and how PUFA-ctS interactions affect the one discrete biochemical function of aS documented to date: inhibiting Phospholipase D. Our focus on a key role for aS in lipid metabolism and membrane vesicle formation/stability derives from a novel set of observations made by the two principal investigators. Moreover, it is strongly supported by recent unbiased genetic screens in aS-expressing yeast or Drosophila that implicated a function of aS in lipid regulation and membrane trafficking. New findings emanating from this grant should simultaneously shed light on the physiology of aS and the earliest steps in its pathological oligomerization, with attendant therapeutic insights.